application of electrochemical surface plasmon resonance
TRANSCRIPT
Application of Electrochemical Surface Plasmon Resonance
(ESPR) to the Study of Electroactive Microbial Biofilms
Journal: Physical Chemistry Chemical Physics
Manuscript ID CP-ART-06-2018-003898.R1
Article Type: Paper
Date Submitted by the Author: 24-Aug-2018
Complete List of Authors: Golden, Joel; US Naval Research Laboratory, Center for Bio/Molecular Science and Engineering Yates, Matthew; U.S. Naval Research Laboratory Halsted, Michelle ; University of Tennessee, Bredesen Center for Interdisciplinary Research and Graduate Education Tender, Leonard; Naval Research Laboratory,
Physical Chemistry Chemical Physics
1
Application of Electrochemical Surface Plasmon Resonance (ESPR) to the Study of Electroactive Microbial
Biofilms
Joel Golden1, Matthew D. Yates
1, Michelle Halsted
2, Leonard Tender
1*
1Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington DC, 20375
2 The Bredesen Center for Interdisciplinary Research and Graduate Education, The University of Tennessee,
Knoxville, TN, 37996
Abstract
Electrochemical surface plasmon resonance (ESPR) monitors faradaic processes optically by the change in
refractive index that occurs with a change in redox state at the electrode surface. Here we apply ESPR to
investigate the anode-grown Geobacter sulfurreducens biofilm (GSB), a model system used to study electroactive
microbial biofilms (EABFs) which perform electrochemical reactions using electrodes as metabolic electron
acceptors or donors. A substantial body of evidence indicates that electron transfer reactions among hemes of c-
type cytochromes (c-Cyt) play major roles in the extracellular electron transfer (EET) pathways that connect
intracellular metabolic processes of cells in an EABF to the electrode surface. The results reported here reveal that
when the potential of the electrode is changed from relatively oxidizing (0.40 V vs. SHE) to reducing (-0.55 V vs.
SHE) and then back to oxidizing, 70% of c-Cyt residing closest to the biofilm/electrode (within hundreds of nm
from the electrode surface) appear to remain trapped in the reduced state, requiring as long as 12 hours to be re-
oxidized. c-Cyt storing electrons cannot contribute to EET, yet turnover current resulting from cellular oxidation of
acetate coupled with EET to the electrode surface is unaffected. This suggests that a relatively small fraction of c-
Cyt residing closest to the biofilm/electrode interface is involved in EET while the majority store electrons. The
results also reveal that biomass density at the biofilm/electrode interface increases rapidly during lag phase,
reaching its maximum value at the onset of exponential biofilm growth when turnover current begins to rapidly
increase.
Introduction
Electroactive biofilms (EABFs) act as electrode catalysts due to their electrode-coupled metabolism
whereby a non-corrosive potentiostatically-regulated electrode serves as the metabolic electron acceptor or
donor.1-8
Central to their catalytic properties are the endogenous extracellular electron transport (EET) pathways
constituent electroactive microorganisms (EMs) use to electrically connect electron consuming or generating
intracellular metabolic processes with the underlying electrode surface. Not strictly catalysts,9 EMs conserve a
portion of the electron energy for growth and maintenance, and in doing so can remain active indefinitely, unlike
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inorganic or ex vivo enzyme electrode catalysts which degrade over time. Synthetic biology offers the promise of
enhancing catalytic properties of innate EM, and transforming non-electroactive microorganisms that perform
desired redox reactions into EM by genetic engineering of EET pathways.10
Envisioned applications of EABF-
facilitated electrode reactions include power generation from oxidation of organic matter (electrogenesis) in raw
waste streams (e.g., sewage) and sediments 11-14
; and fixing CO2 into multi-carbon organic compounds
(electrosynthesis) for use as fuels using renewable sources of electricity as a means to mitigate the impact of
anthropogenic carbon emission on global climate change.8, 15-20
Achieving applications of EABFs at worthwhile scales requires a high-level understanding of the underlying
processes. To this end, electrochemical methods have been primarily used to investigate EET of EABFs. These
include chronoamperometry to characterize the rate of growth and maximum sustained rate of electrode-
dependent metabolic activity 21
; cyclic voltammetry (CV)22
to determine the underlying general mechanism of
catalysis (e.g., Nernst-Monod model23-26
); potential-step27
and temperature-dependent electrochemical gating
measurements8, 28-32
to determine properties of long-distance (multi-cell-length) EET (LD-EET) that occur through
some EABFs (i.e., redox conductivity)27, 29, 31, 33
and to determine if heterogeneous EET (H-EET) across the
biofilm/electrode interface is rate limiting27, 29
; and electrochemical impedance spectroscopy (EIS)34-35
which can
provide information on all of the above.
There is a small but growing body of literature wherein spectroscopic methods are combined with
electrochemical methods which provide deeper insights into the mechanisms of EET. Examples include
absorbance spectroscopy,36-38
which established that the oxidation state of c-Cyt in electrode-grown G.
sulfurreducens biofilms is dependent on the electrode potential in a manner consistent with the role of electron-
transfer reactions among c-Cyt in LD-EET; Raman microscopy33, 38-43
which further indicated the role of c-Cyt in LD-
EET of GSB and in mixed community anode-grown EABFs enriched in Geobacter spp. as well as a possible role of
iron-sulfur clusters in EET of a cathode-grown mixed community electroautotrophic EABF8; florescence
spectroscopy,44
which provided evidence that the multiple non-Nernstian peaks in non-turnover voltammetry of
mixed community anode-grown EABFs enriched in Geobacter spp. (also observed for GSB) may reflect a c-Cyt
involved in H-EET that undergoes an electrode potential-dependent structural change; and most recently
differential electrochemical mass spectrometry,45
which enabled real-time tracking of the electrode potential-
dependent rate of CO2 generation by a mixed community anode-grown EABF enriched in Geobacter spp.,
suggesting that acetate oxidation, which requires EET to the electrode, still persists at a relatively reducing
electrode potential (-0.30 V vs. SHE).
To these combined spectroscopic-electrochemical methods we add electrochemical surface plasmon
resonance (ESPR).46
ESPR is based on surface plasmon resonance (SPR), an optical method that is highly sensitive
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to changes in refractive index that occur with compositional changes at the interface between a medium (e.g., an
aqueous medium) and a conductive surface.47-48
49
SPR is typically used to study adsorption50
and other molecular-
level interactions, such as antibody-antigen binding when one (antibody or antigen) is immobilized on the surface
and the other binds to it.51
The key feature of SPR is its short depth of sensitivity, which drops off exponentially
within 10s to 100s of nanometers from the surface depending on the specific configuration,47
resulting in high
signal-to-noise measurements of interfacial processes. ln ESPR, the conducting surface is simultaneously used as a
working electrode, enabling optical monitoring of electrochemical reactions via the change in the interfacial
refractive index that occurs with the change in oxidation state of redox molecules at the electrode surface.46, 52
This has been established for diffusing redox molecules46
as well as electrode-bound redox molecules,46, 53
including redox polymer-wired enzyme films54
and electrode-bound cytochromes55-56
– the latter two prompting
the work reported on here due to the prevalent role of cytochromes in EET of EABFs including electron transfer
across the biofilm/electrode interface8, 23, 28-29, 38-40, 57-61
as well as similar electrochemical properties of redox
polymer-wired enzyme films and EABFs.23
Here we demonstrate the use of ESPR for the first time to study an EABF, specifically GSB. Changes in the
optical signal intensity is taken here to reflect the sum of 1) changes in biomass density at the biofilm/electrode
interface associated with GSB growth at a fixed electrode potential (0.5 V vs SHE), and 2) changes in oxidation
state of c-Cyt at the biofilm/electrode interface that occur during CV (between 0.40 and -0.55 V vs. SHE). The
results reveal that during lag phase, when current due to EET is negligible, biomass density at the
biofilm/electrode interface increases rapidly, reaching a maximum value at the onset of exponential biofilm
growth when current due to EET begins to rapidly increase.39
Changes in the SPR (optical) signal intensity
observed during CV indicate that at all stages of biofilm growth, as the potential of the electrode is changed from
relatively oxidizing to reducing and then back to oxidizing, approximately 70% of the ESPR detectable c-Cyt appear
to remain trapped in the reduced state, requiring as long as 12 hours to become re-oxidized. This appears to have
no effect on EET as judged by turnover current that is the same before and after CV. This result highlights the dual
role of c-Cyt in EET and electron storage.62
Consequently, only a relatively small fraction of c-Cyt nearest to the
biofilm/electrode interface appear to be involved in or are required for EET to sustain the respiration needs of a
fully-grown stationary phase GSB.
Methods
See Golden et al., 201563
for a detailed description of the SPR system used here. Briefly, a commercial surface
plasmon resonance imager (SPRimager Horizon, GWC Technologies: Kretschmann configuration, 800 ± 6 nm,
manually adjustable fixed angle, CCD detector) was equipped with a chamber to grow anaerobic biofilms. The
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chamber, a 30 ml batch reactor, was designed and fabricated in-house out of poly(methyl methacrylate) (PMMA).
The bottom of the chamber consisted of a replaceable gold-coated commercial SPR substrate (GWC Technologies)
comprised of a SF10 glass slide (25 × 38 × 1 mm) onto which a 7-nm thick titanium adhesion layer was deposited
followed by a 38 nm gold layer. The gold surface faced into the chamber and served as the working electrode.
Before use, each substrate was vigorously rinsed with ethanol followed by sterile DI water and then air-dried. An
electrical connection was made directly to the gold surface near one end of the slide using a low-temperature
soldering iron and indium solder to minimize heat damage to the gold layer. The wired connection was coated
with 5-min epoxy (Devcon) for strain relief. A 0.2-cm diameter (0.0314 cm2) circular electrode was created in the
center of the gold surface by masking the gold surface with biomedical adhesive tape (ARseal 90880, Adhesives
Research, Inc.). We have found that masking the SPR substrate to a relatively small electrode area to chamber
volume ratio minimizes mass transport limitations in the relatively small unstirred chamber, resulting in textbook
voltammetry. The hole was precision-cut using a laser cutter (Mini/Helix 8000 Laser System, Epilog Laser). See
supplemental materials for image of masked SPR substrate and GSB voltammetry. The chamber was sealed to the
SPR substrate with a 2-cm diameter nitrile rubber O-ring. The O-ring was sealed directly against the gold surface
and not the mask. The additional exposed gold area contributed to current and for this reason current and not
current density is reported. The electrical connection to the gold surface was outside the chamber. The top of the
chamber was sealed with a PMMA cover and included openings for a counter (¼”-diameter graphite rod), and
reference electrodes (Ag/AgCl, 3 M KCl, Bioanalytical Systems) and a sparge cannula for maintaining anaerobic
conditions (20:80 CO2:N2). The bottom of the SPR slide was mounted directly on the prism of the SPR imager using
index matching fluid (Cargille Master Calibration Liquid, no. 19268, n = 1.6304). The entire SPR system and growth
chamber were operated within a large incubator at 30oC.
The surface plasmon resonance imager spatially maps, with high sensitivity, changes in refractive index
due to processes occurring within the evanescent field that extends 10s to 100s of nanometers from the electrode
surface. Incident light reflected off the underside of the gold layer (through the underlying glass and prism) is
imaged by a CCD detector, and results in a spatial image of the light intensity reflected by the interface.46, 52, 64-65
Spatial variation in intensity is determined by spatial variation in refractive index at the interface which occurs, for
example, from non-uniform adsorption onto the SPR substrate or use of patterned molecular capture elements. In
the images, brighter regions (higher SPR signal intensity) correspond to a higher refractive index, while darker
regions correspond to lower refractive index. Biomass (proteins, bacterial cells, biofilms, etc.) possesses a higher
refractive index than water.50, 66-67
As such, an increase in SPR intensity is taken here in to indicate and increase in
biomass density at the biofilm/electrode interface. Electrochemical activity also affects SPR intensity46
, as well as
temperature.48
For the specific SPR imager used here, SPR intensity correlates linearly with CCD pixel intensity for
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intensity values between 75 and 185 (maximum range of 0−255, 8 bits). The SPR angle was set within this range
before each experiment. When the intensity exceeds 180, the correlation is still positive, but linearity is lost,
yielding progressively smaller increases in pixel intensity with increasing index of refraction until the SPR intensity
is saturated. In cases where biofilm growth caused pixel values to exceed this range, the SPR angle was reset to be
within the linear range, reducing sensitivity to changes in the interfacial refractive index.
Sequential SPR images were recorded over time to acquire spatiotemporal changes in the interfacial
refractive index during biofilm growth. Images were captured at 20 s intervals during the growth experiments. The
instantaneous SPR intensity depicted in Fig. 1 and Fig. 2 as a function of time and applied potential is the average
SPR pixel intensity of the exposed 2-mm diameter gold electrode at each instance using ImageJ.
The SPR chamber was equipped with an electrochemical reference electrode and a counter electrode,
enabling simultaneous electrochemical studies utilizing the SPR substrate as the working electrode. The working
electrode was set to 0.30 V vs. Ag/AgCl during growth except when performing cyclic voltammetry (CV). CV was
performed every 12 hours whereby the potential of the gold substrate was swept from 0.2 to -0.75 and back to
0.2 V (vs Ag/AgCl) at 1 mV/s, twice. All voltammetric experiments were performed with a software controlled
potentiostat (Gamry 1000E, Gamry Instruments Inc.). See supplemental materials for depiction of ESPR for a
reversible soluble redox molecule (ferrocenemethanol) verifying the ability to perform ESPR by methods
described above.
Established procedures were used to grow Geobacter sulfurreducens biofilms (for example see Yates et
al., 20171, Yates et al. (2016)
28 or Phan et al. (2016)
68). Briefly, G. sulfurreducens colonies were grown on 1.5%
minimum media (NB: nutrient broth) agar plates containing acetate (20 mM), fumarate (40 mM), trypticase
peptone (1 g/L) and cysteine (1 mM) and incubated in an anaerobic chamber. Colonies were then picked and
placed into sterile, anaerobic NB medium (g/L: 0.38 KCl, 0.2 NH4Cl, 0.069 NaH2PO4·H2O, 0.04 CaCl2·2H2O, 0.2
MgSO4·7H2O, 2 NaHCO3; 10 ml/L trace minerals; final pH 6.8) containing acetate (20 mM) and fumarate (40 mM)
and incubated at 30°C for two days until the OD600 reached ca. 0.5. The SPR chamber was then inoculated with 3
ml (10% inoculum) of the cultured medium. The medium used in the reactor was the same as the culture medium
except fumarate was excluded, and 10 mM acetate was added.
Results
Biomass density at the GSB/electrode interface.
Biomass density at the GSB/electrode interface, indicated by SPR signal intensity, increases rapidly during
lag phase (period of biofilm growth following inoculation marked by low current). It reaches a maximum value at
when current due to acetate oxidation, coupled with EET to the electrode surface (poised at 0.50 V vs SHE), begins
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to rapidly increase (Fig. 1, and Fig. S7 in supplemental material for biological replicates). The simultaneously
recorded current (Fig. 1) exhibits the characteristic stages of GSB biofilm growth.61
Here, SPR intensity (CCD
detector pixel intensity) indicates the intensity of light (800 nm) reflected by the gold electrode. The balance of
incident light is absorbed by excitation of a surface plasmon along the biofilm/electrode interface and not by c-
Cyt, which absorb between 350 and 600 nm.36-37
Plasmon excitation is dependent on the interfacial refractive
index, which increases with increasing biomass density at the biofilm/electrode interface, resulting in an
increasing SPR intensity as observed here. For the specific ESPR experiment depicted in Fig. 1, the incident angle
was adjusted 4 days after inoculation in order to maintain a linear response to changes in refractive index, which
occurs for SPR intensities between 75 and 185 for the specific instrumentation used here (no attempt was made
to limit biofilm growth so as to remain in the linear response region without changing the incident angle). As such,
the decrease in SPR intensity that occurs after the angle change suggests that a relatively large decrease in
biomass density at the biofilm/electrode interface occurred during exponential growth. For comparison, in
biological replicates in which the angle was not changed (Fig. S7), SPR intensity rises as in Fig. 1, converging to a
fixed value by the onset of exponential growth. In those measurements however, the SPR intensity increased well
beyond the linear response range and may not have been responsive to a subsequent decrease in interfacial
biomass density. Regardless, in all cases the SPR intensity rose during lag phase reaching its maximum value by
the onset of exponentially rising current. A control in which the inoculum contained heat-killed dead cells (55 °C
for 4 hours) induced a mitigated response (Fig. S4), indicating that the SPR response observed in Fig. 1 can be
primarily attributed to biological activity of cells at the electrode surface (e.g., adhesion). The SPR response
observed here is consistent with that observed for the initial period of growth of E. coli biofilms monitored by
SPR,69
in which an initial rise in SPR intensity was attributed to displacement of water by cell replication at the
interface. The response is also consistent with previous results indicating that during lag phase, GSB growth
proceeds by cell replication resulting in formation of tight-packed single-cell thick domains that expand across the
electrode surface before the onset of exponentially rising current at which time multiple cell layers begin to
form.39
Abiotic ESPR voltammetry
Fig. 2 depicts a sequence of ESPR cyclic voltammograms (CVs) recorded at different stages of growth for
the same GSB depicted in Fig. 1 where the potential dependency of current is consistent with CV recorded during
biofilm growth25
(see Fig. S2 for the same data as well as biological replicates in which current and SPR signal
intensity are plotted vs. potential). Fig. 2a was recorded just prior to inoculation and is attributed to reduction of
trace oxygen in the SPR chamber. Two CVs were recorded consecutively and the magnitude of the cathodic
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current during the second CV is lower than the first, consistent with oxygen depletion during the first CV resulting
in reduced mass transport of oxygen to the electrode during the second CV. The SPR signal intensity tracks both
the potential and current as expected for an electrochemical reaction.46, 52-53, 64, 70-71
Most notable is that the SPR
intensity observed at the beginning of the first CV and the end of the second CV are nearly the same, indicating
that performing the CVs had no lasting effect on composition or redox state of the interface. In addition, the
minimum SPR signal intensity observed toward the middle of each anodic scan are the same and lags behind the
current minima, emphasizing that in the case of a mass transfer-limited electrochemical reaction (the case here),
current is dependent on the concentration gradient of the redox species (oxygen) at the electrode surface, which
is expected to reach a maximum value then decrease during CV due to depletion of the redox species near the
electrode surface72
; whereas SPR signal intensity is dependent on the concentrations of oxidized and reduced
forms of the redox species at the electrode surface which tend toward limiting values.46
Biotic ESPR Voltammetry
Fig. 2b depicts two turnover CVs consecutively recorded during lag phase 12 hours after inoculation for
which it is assumed that residual oxygen was mostly drawn down by cells in the chamber. Here current and SPR
intensity are attributed to the nascent GSB biofilm. Unlike Fig. 2a, the current minima of the consecutive CVs are
the same indicating no depletion effects (consistent with excess acetate) in the medium. While the SPR intensity
decreases with a decreasing anodic current, the SPR intensity minima of the second CV is lower than the first, and
SPR intensity is significantly lower at the end of the second CV than at the start of the first CV. We have observed
that it takes as long as 12 hours for the SPR intensity to recover to its initial value just before CVs were performed
(Fig. 3). In contrast, current recovers almost immediately (Fig. 3). The same trends are observed at early
exponential phase (Fig. 2c), at mid exponential phase (Fig. 2d), and at stationary phase (Fig. 2e). Fig. 2f depicts the
same trend in which SPR intensity at the end of the second CV is significantly lower for the stationary phase GSB
depicted in Fig. 2e, but under non-turnover condition (acetate-free medium) where the suppression in SPR signal
intensity is more pronounced.
DISCUSSION
It is established that c-Cyt of GSB play a central role in EET including electron transfer across the
biofilm/electrode interface42,43
and that c-Cyt oxidation state is linked to the electrode potential in a near-
Nernstian manner centered on the turnover CV midpoint potential.23-24, 26, 36-37
It is also established that the
change in the oxidation state of an electrode-bound cytochrome during CV (sans biofilm) can be monitored by
SPR where the SPR signal is fully reversible (i.e., the cytochrome is not trapped in either oxidation state beyond a
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fraction of a second).56, 64
We therefore contend that the decrease and increase in SPR signal intensity centered on
the CV current midpoint potential for each cathodic and anodic scan depicted in Fig. 2b-f, result from reduction
and oxidation of c-Cyt at the biofilm/electrode interface in response to the changing electrode potential.
Moreover, we contend that the overall downward trend in SPR signal intensity from the beginning to the end of
the two sequential CVs results from storing electrons (i.e., rectification)73
by a portion of c-Cyt at the interface
that persist in the reduced form. To be involved in EET, c-Cyt must convert rapidly between the oxidized and
reduced forms29
. We also contend therefore that c-Cyt involved in storing electrons are not involved in EET
because they are not immediately re-oxidized during the anodic scans and because the magnitude of
turnover/non-turnover current is the same just before and after performing the CVs.
Following Shan, et al. (2010)46
, SPR signal intensity associated with a redox molecule is a linear sum of
contributions attributed to the oxidized and reduced forms. For the SPR measurements reported here, the
incident angle was fixed such that the SPR signal correlates linearly for CCD pixel intensity between 75 and 185. As
such, for each growth phase depicted in Fig 2., assuming that c-Cyt at the biofilm/electrode surface are fully
oxidized at the beginning of the first CV for which the SPR signal is greatest, and fully reduced when the SPR signal
is at its lowest value during the second CV, the fraction of c-Cyt in the oxidized state changes linearly with the
change in SPR signal between the maximum and minimum values. Since the SPR signal intensity only recovers by
approximately 30% by the end of the second CV at each stage of growth, approximately 70% of c-Cyt detected by
SPR are trapped in the reduced state under turnover condition at each stage of growth. Under non-turnover
condition, approximately 95% of c-Cyt are trapped in the reduced state. If c-Cyt at the cathodic peak of the second
CV are not fully reduced, then the fraction of c-Cyt involved in electron storage is even greater. Taken together,
the results depicted in Fig. 2 suggest that beginning soon after inoculation, the majority of ESPR detectable c-Cyt
(those residing within 100 nanometers from the electrode surface) store electrons rather than participate in EET.
The stored electrons may originate from the backflow of low-potential electrons from the electrode when at a
sufficiently low potential. Both turnover and non-turnover CV of GSB exhibit non-negligible cathodic current at
low potentials and there is precedence for GSB to use an electrode as an electron donor74
. Alternatively, the
stored electrons may be high potential electrons resulting from persistent acetate oxidation, where the electrode
cannot accept high potential electrons when it is at a low potential. In addition to the abiotic CVs (Fig. 2a), which
did not exhibit a net decrease in SPR intensity, we also performed ESPR of ferrocenemethanol (Fig. S3) using the
same instrumentation. Here too, the SPR intensity at the start and end of two consecutive CVs was the same,
indicating that the observed decrease in SPR intensity for GSB is not an artifact of our instrumentation. Moreover,
it is established that the SPR signal intensity of electrode-bound cytochromes56, 64
does not change appreciably
between the start and end of CVs, indicating that the effect observed here is not a generic cytochrome effect.
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We cannot rule out that the slow relaxation of the SPR signal reflects persistent structural changes that
occur at the biofilm/electrode interface and are induced by a low electrode potential. Such changes could result,
for example, by electrostatic repulsion among reduced c-Cyt accumulating at the interface. Surface enhanced
resonance Raman spectroscopy (SERRS) however revealed that for a mixed community EABF enriched from
wastewater (generally a reliable source of Geobacter spp.) exhibited Geobacter-esque voltammetric features42
, as
much as 90% of c-Cyt residing within 7 nm of the electrode surface remain reduced regardless of the electrode
potential for at least 18 seconds. The slow relaxation we observe here however was not considered in that study.
Conclusions
It is established that GSB accumulates electrons in c-Cyt toward the outer surface of the biofilm.33, 36-37, 40
The
results reported here suggest that electrons may also accumulate in c-Cyt right at the electrode surface requiring
as long as 12 hours to be discharged. This may have escaped previous detection by Raman microscopy33, 39-40
and
absorbance spectroscopy.36-37
While these methods are highly sensitive to c-Cyt oxidation state, they lack the high
sensitivity of ESPR to processes localized at the electrode surface. As such, while c-Cyt involved in electron storage
may dominate the biofilm/electrode interface, they may comprise a tiny fraction of total c-Cyt associated with the
biofilm.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This was work supported by NRL base funds and by the Applied Research for the Advancement of S&T Priorities
(ARAP) Program Proposal: Joint Services Laboratories' Capabilities in Synthetic Biology for Military Environments
(SBME). Student research (MH) was sponsored by the Office of Naval Research NREIP program.
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Figures
Figure 1. ESPR recorded during GSB growth while maintaining the electrode at 0.30 V vs. Ag/AgCl (0.50 V vs. SHE),
turnover current (black) and SPR signal (gray). Spikes due to recording CV every 12 h. At 4 days the SPR incident
angle was reset. Biofilm growth states indicated above vertical axis. See supplemental materials for biological
replicates. Letters a-e indicate time when CVs depicted in Fig. 2 were recorded.
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Figure 2. CVs at different growth phases depicted in Figure 1. Two CVs were performed consecutively from 0.20 V
to -0.75 V vs. Ag/AgCl (0.40 to -0.55 V vs. SHE) at 1 mv/s. a) abiotic, b) lag phase, c) early growth, d) mid growth, e)
stationary phase, f) non-turnover. See supplemental materials for biological replicates.
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Figure 3. Representative chronoamperometry data from Figure 1 (magnified) comparing recovery of the SPR
signal (gray) to turnover current (black) after performing two consecutive CVs at 132 h, 144 h and 156 h. Turnover
current (recovers almost immediately whereas the SPR signal takes as long as 12 hours to recover.
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Page 16 of 17Physical Chemistry Chemical Physics
Graphical abstract (Table of contents)
Electrochemical Surface Plasmon Resonance (ESPR) of an electrode-grown Geobacter sulfurreducens
biofilm indicating that when the potential is swept from reducing to strongly oxidizing, as much as 70%
of cytochromes residing within hundreds of nanometers from the electrode surface remain trapped in
the reduced form. This does not effect on the ability of the biofilm to transfer its respired electrons to
the electrode surface.
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